Protein--protein interaction is one of the mechanisms of signal transduction processes. One such process involves non-receptor type protein-tyrosine kinases (PTKs) of the Src family, which signal in normal and transformed cells. In recent years, much of the attention has been concentrated on certain specific regions of PTKs, in particular three structural domains, termed SH2 (SH stands for Src homology), SH3, and PH (pleckstrin homology). In addition to the Src family of proteins, these domains are present in a wide variety of proteins implicated in signal transduction processes.
The yes proto-oncogene encodes a member of the Src family of non-receptor type protein tyrosine kinases (PTKs) (Cooper, 1990; Sudol, 1993). The Src family kinases have been implicated in signal transduction processes because they physically associate with certain membrane receptors and functionally respond to the binding of cognate ligands or receptor crosslinking (Bolen, 1991). The functional response of receptor-associated PTKs is usually manifested by an increase in tyrosine phosphorylation of cellular proteins (Bolen, 1991). The Yes protein kinase was shown to be functionally associated with platelet-derived growth factor receptor in fibroblasts Kypta et al., 1990), with glycoprotein IV (CD36) in platelets (Huang et al., 1991), and with the high-affinity IgE receptor in mast cells (Eiseman & Bolen, 1992), but further signaling steps through these complexes are not known.
Another clue pointing to the involvement of the Src family of PTKs in signaling processes comes from the identification of structural domains, termed SH2 and SH3 (SH for Src homology), which are present in the amino-terminal half of Src family members and are also found in a wide variety of proteins implicated in signal transduction processes (Margolis, 1992; Pawson & Gish, 1992). The SH2 domains are known to interact specifically with phosphotyrosine-containing proteins (Pawson & Gish, 1992; Birge & Hanafusa, 1993; Pawson & Schlessinger, 1993) and the resulting complexes are involved in signal transduction events initiated by PTKs (Cantley et al., 1991). The SH2 domain of Src PTKs is involved in substrate recognition and in the regulation of kinase activity by maintaining a repressed conformation of PTKs (Kanner et al., 1991; Roussel et al., 1991). The precise role of SH3 domains in signal transduction has yet to be completely elucidated (Musacchio et al., 1992a; Mayer & Baltimore, 1993; Pawson & Schlessinger, 1993) but the accumulating genetic, biochemical, and structural data implicate these domains in mediating noncovalent protein-protein interactions essential for cellular and intercellular signaling (Clark et al., 1992; Musacchio et al., 1992b; Yu et al., 1992; Booker et al., 1993; Kohda et al., 1993; Koyama et al., 1993; Li et al., 1993; Noble et al., 1993; Rozakis-Adcock et al., 1993; Ren et al., 1994; Feller et al., 1994). Studies with the SH3 domains of the Ab1 kinase and the Grb2 adaptor protein identified a 10 amino acid long proline-rich motifs that are present in the proteins that bind to the SH3 domains and mediate the protein--protein interaction (Cicchetti et al., 1992; Li et al., 1993; Ren et al., 1993; Rozakis-Adcock et al., 1993; Williamson, 1994; Yu et al., 1994). For Src and other members of the family, it is presumed that binding of specific proteins to their SH3 domains may result in the modulation of their enzymatic activity and thus could be a part of the signaling mechanism by cellular and oncogenic forms of the Src Emily PTKs (Kato et al., 1986; Potts et al., 1988; Nemeth et al., 1989; Hirai & Varmus, 1990; Seidel-Dugan et al., 1992; Wages et al., 1992; Cooper & Howell, 1993; Liu et al., 1993). It has also been reported that SH3 domains of Src PTKs interact with substrates (Kanner et al., 1991; Seidel-Dugan et al., 1992; Liu et al., 1993) and with other signaling molecules including unknown serine and/or threonine kinases (Weng et al., 1993).
Our functional studies of the Yes proto-oncogene started with the generation of polyclonal antibodies directed to the bacterially expressed fusion protein corresponding to the unique and SH3 domains of Yes (Sudol & Hanafusa, 1986). Interestingly, the resulting antibody showed strong immunoreactivity with the SH3 domain and weaker reaction with the unique domain. Based on this observation we used the original anti-Yes IgG to generate polyclonal anti-idiotypic antibodies (Jerne, 1974) expecting a reagent that would mimic a conformation of the SH3 domain of Yes and would allow us to isolate Yes binding proteins.
We report here the identification, characterization, and cDNA cloning, of a novel protein that binds to the SH3 domain of the Yes proto-oncogene product. Anti-idiotypic antibodies were used to identify the protein and to clone its cDNA from an expression library. The presence of serine phosphorylation along with a proline-rich motif involved in SH3 binding implicates YAP65 in signaling processes. It is possible that interaction between Yes and YAP65 represents a novel link between pathways transduced by protein-tyrosine and serine kinases.
A novel Yes-associated protein (YAP) of 65 kDa was identified in chicken by one of the inventors herein (Sudol, 1994, "Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product," Oncogene 9:2145-52, which is incorporated herein by reference in its entirety, and which corresponds to the first Example disclosed infra).
References cited herein only by author and year are listed at the end of the specification, after the last example. The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.